This invention relates to a method for analyzing catecholamines contained in living body fluid, particularly to a method for analysis capable of treating catecholamines while maintaining them in a stable state and to a sample preparation liquid suitable for stabilization of catecholamines.
Catecholamine is a generic name for norepinephrine (NE), epinephrine (E), dopamine (DA), etc. and plays an important role as a neurotransmission substance or an adrenal medulla hormone. In order to study the functions of sympathetic nerves and adrenal medulla such as hypertonia, pheochromocytoma, sympathetic neuroblastoma, and the like, it is important to determine quantitative and qualitative changes of catecholamines in a living body sample such as blood, urine, etc.
In general, living body samples such as blood, pith liquid, etc., contain a higher concentration of protein and a lower content of catecholamines as compared with urine. Therefore, analysis of catecholamines in blood or pith liquid particularly requires a method of higher sensitivity than the methods conventionally used for analysis of catecholamines in urine. Moreover, due to the high protein concentration in blood or pith liquid, it is required to prevent the interference with the analysis by proteins. Furthermore, when the analysis is carried out by liquid chromatography, there is a problem of shortening of the life of the column therefor due to the high protein concentration.
In order to prevent the interference with the analysis by proteins and the deterioration of the column for liquid chromatography, it is general practice to carry out deproteinization treatment of a living body sample such as treatment with perchloric acid before liquid chromatography analysis. However, catecholamines are very unstable in neutral and alkaline conditions, and easily undergo a change such as oxidation. Since living body samples such as blood, pith liquid, etc., have a lower content of catecholamines than urine, the deproteinization treatment remarkably damages the catecholamines, so that it has not been possible to obtain any satisfactory analysis accuracy. It is known that catecholamines can be stabilized to some extent by adding an antioxidant such as EDTA, ascorbic acid, sodium thiosulfate, dithiothreitol, or the like to a living body sample and preserving the sample at not higher than -20.degree. C. However, the procedures therefor are made complicated, which complication has been an impediment to automation of catecholamine analysis. The foregoing matters have been discussed in Analytica Chimica Acta, 165 (1984), pp. 171-176 and "Rinsho Kensa (Clinical Examination)" 32, 12 (1988), pp. 1,522-1,527.
Catecholamines are determined generally by high-performance liquid chromatography. The assay thereof is carried out by fluorometry or an electrochemical method. The electrochemical method is susceptible to be influenced by the other components, and it is therefore not suitable for analysis of a trace amount of catecholamines. The fluorometry is classified into a DPE method using diphenylethylenediamine and THI method using trihydroxyindole. In the THI method, DA detection sensitivity is low, and catecholamines, e.g. in the blood cannot be analyzed. Therefore, the DPE method is the most suitable for analysis of a trace amount of catecholamines.
However, the optimum pH for a labeling reaction in the DPE method is pH 6.0 to 8.0, particularly in the vicinity of pH 7.0. In order to obtain high sensitivity, therefore, there is a dilemma in that the pH of the reaction solution prepared by mixing a sample with a labeling agent containing DPE is required to be brought close to the optimum pH, particularly to the vicinity of pH 7.0, whereas catecholamines are unstable under a pH around 7.0.
As the deproteinization method in the analysis of plasma catecholamines by the precolumn fluorescence HPLC method using DPE, studies have been made on ion-exchange column method which utilizes the difference in polarity between the proteins and the intended component for separating them, perchloric acid method which uses the precipitation of proteins by modification of the proteins with an acid, ultrafiltration method which uses the difference in size of molecule for the separation, and alumina column method which uses a column capable of selectively adsorbing catecholamines. However, the recovery ratios of these deproteinization methods are: 85% for ion-exchange column method, which shows the highest recovery ratio; 70 to 80% for perchloric acid method and ultrafiltration method; and 60 to 70% for alumina column method. Thus, the recovery ratios of these methods are all less than 90%. Therefore, these methods are unsatisfactory in their poor reproducibility and inability to carry out high-accuracy analysis. The foregoing matters are discussed in Journal of Chromatography, 344 (1985), pp. 61-70.
The above conventional techniques have not considered the stability of catecholamines during the preparation or analysis of the sample containing proteins to be removed. Nor have the above techniques considered the difficulty in controlling the pH of the reaction solution due to the difference between the optimum pH in the case of using the DPE method as a detection method and the pH at which catecholamines are stable. As a result, the above conventional techniques have a problem in the poor recovery ratio through the deproteinization and the reaction as a whole.